The long-term goals of this project are to understand how extremely alkaliphilic Bacillus strains (non-halophiles) achieve respiration-coupled ATP synthesis and cytoplasmic pH regulation. These two central alkaliphile functions are unique examples of proton-coupled energetic processes in these extremophiles. Other bioenergetic work is Na+-coupled. ATP synthesis and pH homeostasis proceed robustly under conditions that challenge strictly chemiosmotic mechanisms requiring inward proton translocation. The specific aims will take advantage of newly developed experimental approaches to alkaliphilic Bacillus firmus OF4, and include the following. (1) A comprehensive transposon- mediated screen for non-alkaliphilic mutants may reveal undiscovered gene products that are related to the akaliphile's resolution of its energetic dilemma. Such mutants or the genes discerned would then be used in the other studies together with targeted mutations already planned. (2) Site-directed mutations and gene replacements of "alkaliphile-specific sequence motifs" in F1Fo- ATPase (atp genes) will clarify their importance in alkaliphile ATP synthesis. In part, these studies test models for oxidative phosphorylation in which protons emerging from (a) respiratory chain complex(es) reach the ATP synthase via direct protein-protein interactions. The protein-protein interactions themselves will be examined in an expanded array of approaches, including in vivo use of complexes tagged with green fluorescent protein variants. (3) Potentially novel features of two Na+/H+ antiporters that are involved in pH homeostasis, MrpA and NhaC, will be examined in a combination of biochemical and molecular approaches to clarify their possible relationship to the capacity of the alkaliphile for using antiporters to establish and maintain a transmembrane pH gradient that is greater than 2 units more acid inside than out while retaining a large transmembrane potential, oriented conventionally, i.e. positive out. (4) Another solution to the bioenergetic problems of both pH homeostasis and ATP synthesis may be reliance upon effective retention of protons on the membrane surface after their emergence from the respiratory chain. The protons might then reach the antiporters and ATP synthase without equilibrating with the bulk alkaline medium. The role of a newly identified S-layer protein and, in particular, of an "excess of carboxylates" on outer loops of alkaliphile membrane proteins will be examined using targeted mutagenesis to disrupt, respectively, the sap gene (encoding the S-layer protein) and the putative carboxylate network on selected "proton-consumers" such as MrpA or ATP synthase and perhaps on the cytochrome oxidase. Special adaptations used by the extremophile to successfully meet the energetic challenges of ATP synthesis and pH homeostasis will clarify the adaptive "plasticity" available to chemiosmotic mechanisms. The adaptations are also likely to include mechanisms that are widely used but not yet recognized in other cells or organelles that are under specific pressure to maximize proton-coupling efficiency. The antiporter studies per se are of general interest inasmuch as homologues of those involved in alkaliphile pH homeostasis are widespread among prokaryotes. NhaC increasingly appears to represent a new example of the convergence of monovalent cation/proton antiporters with mechanisms of antibiotic-resistance, i.e. as a multifunctional antiporter. MrpA may represent another trend that could emerge in energy-constrained contexts, i.e. the capacity for a secondary antiporter to adopt a primary type of energization in the proper protein complex or vice versa.